Human respiratory syncytial virus (RSV) causes respiratory tract infections. It is the major cause of hospital visits during infancy and childhood. After translation of viral mRNAs, a full-length (+) antigenomic RNA is produced as a template for replication of the (−) RNA genome. Infectious recombinant RSV (rRSV) particles may be recovered from transfected plasmids. Co-expression of RSV N, P, L, and M2-1 proteins as well as the full-length antigenomic RNA is sufficient for RSV replication. See Collins et al., Proc Natl Acad Sci USA., 1995, 92(25):11563-11567 and U.S. Pat. No. 6,790,449.
Despite the existence of methods of generating RSV particles from cloned cDNA, stability of RSV cDNA remains a challenge. A region of the RSV small hydrophobic protein (SH) gene is unstable as cloned cDNA. See e.g., Skiadopoulos et al., Virology 345, 492-501 (2006). Investigators have suffered failures in cloning RSV cDNA in plasmids, despite extensive experience with other viruses and cDNA cloning. Labs typically use a RSV antigenomic cDNA cloned in the plasmid pBR322. In order to maintain the antigenomic cDNA in this plasmid, one typically grows the bacteria at 30° C. and low aeration. Nevertheless, plasmids frequently undergo rearrangements and clone loss. Taken together, plasmid stability is a factor limiting progress in RSV research and vaccine development. Thus, there is a need to identify improved methods of generating RSV.
One may recover viruses from bacterial artificial chromosome (BAC) vectors. See Roth et al., Vet Res., 2011, 42(1):3 and Alder et al., Rev Med. Virol., 2003, 13(2):111-21 and U.S. Pat. No. 7,892,822. BAC recombineering refers to a method of introducing mutations in cDNAs cloned in a BAC vectors via homologous recombination in E coli. A BAC recombineering system based on selection and counter-selection of the galK operon was disclosed by Warming et al, Nucleic Acids Research, 2005, 33, e36. References cited herein are not an admission of prior art.